5alpha-hydroxylation of a-nor-5alpha-steroids



United States Patent 3,226,303 Sa-HYDROXYLATHUN 0F A-N0R-50t-TERQHDAllen I. Laskin, Somerset, Samuel C. Pan, Metnchen, and

Frank L. Weisenborn, Somerset, N.J., assignors to Ulin MathiesonChemical (Iorporation, New York, N.Y., a corporation of Virginia N 0Drawing. Original application Jan. 31, 1963, Ser. No. 255,180. Dividedand this application Mar. 31, 1965, Ser. No. 449,673

Claims. (Cl. 19551) This application is a division of our application,Serial No. 255,180, filed January 31, 1963.

This invention relates to, and has for its objects the provision of amicrobial method for producing SOL-hydroxy steroids of the A-nor series,saturated in the A and B rings; new intermediates prepared thereby; andprocesses for converting said intermediates to 3,5-dehydro steroidderivatives.

It has been found that steroids of the A-nor series, that are saturatedin both the A and B rings and contain a Set-hydrogen substituent can beconverted to 5u-hydroxyl derivatives by subjecting them to the action ofenzymes of Cokeromyces recurvatus or to the action of the organismitself, under oxidizing and preferably aerobic conditions.

Among the steroids which may be hydroxylated by the practice of thisinvention are those steroids which are members of theA-nor-5ot-androstane series and, preferably, the A-nQr-Su-pregnaneseries. Particularly preferred are steroids of the2-keto-A-nor-5a-pregnane series (i.e. 2-keto-A-norallopregnane series),such as A-nor-Sapregnane-2,20 dione, A-IlOf-Srx pregnane-Z-oneZOfi-oland Zoe-acetoxy-A-nor-Sa-pregnane-Z-one.

The nature of the product will depend on the steroid substrate chosen.In all cases a 5a-hydroxy steroid is formed. If, however,A-nor-5a-pregnane-2-one-20fi-ol is selected as the steroid, in additionto the 5a-hydroxylation, the 17-side chain is removed andA-nor5a-androstane-5a,l7,8-diol-2-one is obtained as the major product.

The Swhydroxy steroids can then be dehydrated, according to anotherprocess of this invention to yield the known 3,5-dehydro steroidderivatives.

The action of the enzymes of Cokeromyces recurvatus to produce theSa-hydroXy steroids can be utilized either by including the steroid inan aerobic culture of the microorganism, or by bringing together, in anaqueous medium, the steroid, air and enzymes of non-proliferating cellsof the microorganism.

In general, the conditions of culturing the Cokeromyces for the purposesof this invention are (except for the inclusion of the steroid to beconverted) the same as those of culturing various aerobic microorganismsfor the pro duction of antibiotics, i.e., the microorganism isaerobically grown in contact with (in or on) a suitable fermentationmedium. A suitable medium essentially comprises a source of nitrogenousand growth-promoting factors, and an assimilable source of carbon andenergy. The latter may be a carbohydrate and/or the steroid itself.Preferably, however, the medium includes an assimilable source of carbonand energy in addition to the steroid.

The nitrogen source materials may be organic (e.g., soybean meal,cornsteep liquor, yeast extract, meat extract and/or distillerssolubles) or synthetic (i.e., composed of simple, synthesizable organicor inorganic compounds such as ammonium salts, alkali nitrates, aminoacids or urea).

As to the source material, lipids, especially (1) fatty acids having atleast 14 carbon atoms, (2) fats or (3) mixture thereof, may be used.Examples of such fats are lard oil, soybean oil, linseed oil, cottonseedoil, peanut oil, fancy mutton tallow, sperm oil, olive oil, tristearin,tripalmitin, triolein and trilanrein, and illustrative fatty acidsinclude stearic, palmitic, oleic, linoleic and myristic acids.

Other carbon-containing materials may also be used. For example, suchmaterials as glycerol, glucose, fructose, sucrose, lactose, maltose,dextrins, starches, whey, etc., are adequate carbon source materials.These materials may be used either in purified state or as concentrates,such as whey concentrate, corn, wheat or barley mash; or mixtures of theabove may be employed. It is to be noted, however, that the steroid isadded to the fermentation medium essentially as a precursor and not asan energy source.

The steroid products formed are then separated from the culturechromatographically as more fully detailed in the following examples.

The isolated 5u-hydroxy steroids can then be converted to theirrespective 3,5-dehydro derivatives by dehydration, preferably byemploying a basic substance, such as potassium t-butoxide, potassiumhydroxide, sodium methoxide, and the like. These 3,5-dehydro steroidsare known substances.

The following examples are illustrative of the invention (alltemperatures being in degrees Centigrade):

EXAMPLE 1 5 ot-hydroxy-A -1z0r-5a-pregnane-2,2O-dione (A) "FERMENTATIONA stock culture of Cokeromyces recurnatus (Centralbureau voorSchimmel-cultures, Baarn, Holland) is maintained on a Gould agar slant,the slant containing as a nutrient medium (A) Yeast extract 2.5

K HPO 1 Glucose 10 Agar 20 Water to make 1 liter. (Sterilized at 121 for30 minutes.)

The surface growth on such slant, which has been incubated at for twoweeks is suspended in 0.01% Duponol solution. This suspension is used toinoculate two 250 ml. Erlenmeyer flasks, each containing 50 ml. of thefollowing medium (B) G. Malt cereal extract 10 Wilsons peptone No. 15920 Starch 20 Cerelose 44 NaNO 3 KH PO 1 KCl 0.5 MgSO, 7H O 0.5 FeSO -7HO 0.02

Tap water to make 1 liter. (Sterilized at 121 for minutes; pH adjustedto 7.0.)

After incubating at 25 on a rotary shaker set at 280 rpm. and a 2"stroke for 96 hours, ml. of the fully grown culture is used to inoculateeach 4 liter Erlenmeyer flask, containing 400 ml. of the same medium(B). To each flask is added 2.0 ml. of a 6% (w./v.) solution ofA-nor-5a-pregnane-2,20 dione in dimethyl forrnamide, which has beensterilized by filtration through sintered glass thereby giving a finalconcentration of 300 g. of steroid per ml. of medium. These flasks arethen incubated for seven days under the same conditions used to incubatethe 250 ml. Erlenmeyer flasks.

3 (B) ISOLATION OF 5ll-HYDROXY-A-NOR-5 Z-PREGNANE- 2,'20DION E Thecontents of the flasks obtained in step (A) are pooled and extractedtwice with its volume of chloroform. The chloroform extract is washedtwice with /s its volume of water, dried over anhydrous sodium sulfateand evaporated to dryness. The dried residue is taken up in a smallvolume of 1:1 methanol-chloroform and chromatographed on thin layerplates. Silica Gel-G (E. Merck, Germany) with plaster of Paris as binderspread to a thickness of 0.5 mm. is used as the adsorbent and a mixtureof 1 vol. of chloroform and 2 vol. of ethyl acetate is used as thedeveloping solvent. An amount of the extract representing 20 mg. of thesteroid substrate is applied per inch of the plate width. The solventfront migrates -20 cm. in 90 minutes. After the solvent evaporates off,the plate is exposed to iodine vapor for one hour. The steroids appearas brown bands. These bands are marked out with a stylus. Afterevaporating off the iodine by leaving the plate exposed in a wellventilated hood overnight, these bands are eluted with a 1:1methanol-chloroform mixture. The methanol-chloroform eluate of the bandat Rf=0.65 is evaporated to dryness. The residue is partitioned betweenequal volumes of chloroform and 50% (v./v.) methanol in water. Thechloroform phase is washed twice with water and dried over anhydroussodium sulfate. Evaporation of the chloroform yields crude crystals of5a-hydroxy-A-nor- 5u-pregnane-2,20-dione, which are recrystallized fromacetone-n-hexane to yield the pure product, M.P. about 242*244. [a] +238(chloroform).

EXAMPLE 2 5 0a,] 7B-Dihydr0xy-A -n0r-5a-andr0stan 6-2-0116 (A)FERMENTATION Following the procedure of Example 1, step (A), a (v./v.)transfer is made from the fully grown culture in the first stage 250 ml.Erlenmeyer flasks to 250 ml. Erlenmeyer flasks, each containing 50 ml.of the same medium (B). After incubating for 48 hours under the sameconditions used in the first stage, 100 ml. of the culture obtained inthe second stage is added to 900 ml. of sterilized distilled water in a4 liter Erlenmeyer flask. To the flask is then added 2.0 ml. of a 6%(W./v.) solution of ZOE-hydroxy-A-nor-Sa-pregnane-Z-one in dimethylformamide which has been sterilized by filtration through sinteredglass, thereby giving a final concentration of 200 ,ug. of steroid perml. of medium. The flask is then incubated for seven days at on areciprocating shaker set at 110 cycles per minute and a 1" stroke.

(13) ISOLATION OF 5a,=17BDIHY DROXYA-NOR-5a- AN'DROSTAN'E-fZ-ONE Thecontents of the flask obtained in step (A) is extracted twice with itsvolume of chloroform and the chloroform extract is then treated asdescribed in step (B) of Example 1. The methanol-chloroform eluate ofthe band at Rf=0.45 is evaporated to dryness. The residue is partitionedbetween equal volumes of chloroform and (v./v.) methanol in water. Thechloroform phase is washed twice with water and dried over anhydroussodium sulfate. Evaporation of the chloroform yields crude crystals of5m,17fi-dihydroxy-A-nor-a.-androstane-Z-one, which are recrystallizedfrom acetone-nhexane to yield the pure product, M.P. about 25l253; [06]+115 (acetic acid).

In a similar manner, A-nor-Sa-androStane-2,20-dione is converted to5ot-hydroxy-A-nor-5a-androstane-2,20- dione.ZOB-acetoxy-A-nor-5a-pregnane-2-one is converted to5a,11-dihydroxy-Z0;?-acetoxy-A-nor-5a-pregnane-2-one and511,1S-dihydroxy-ZOQ-acetoxy-A-nor 50tpregnane-Z-one. In thefermentation of A-nor-5ot-preg nane-2,20-dione, some50c,l700,20fitril1yd10Xy-An0I-5apregnane-Z-one is also formed.

EXAMPLE 3 A-nOr-progesterone 25 mg. ofSat-hydroxy-A-nor-5a-pregnane-2,20-dione is dissolved in 10 ml. oft-butyl alcohol containing mg. of potassium t-butoxicle. After twominutes the solution is neutralized with acetic acid, evaporated todryness and the residue distributed between chloroform and 5% sodiumbicarbonate solution. The chloroform layer is washed with water, driedover sodium sulfate and concentrated to dryness. The product isrecrystallized from acetone-hexane to give about 12 mg. of pureA-norprogesterone, MP. 152l54.

EXAMPLE 4 A mar-testosterone 20 mg. of 5a,l7,8dihydroxy-A-nor-5a-androstane 2- one is dissolved in 8 ml. of t-butylalcohol containing 80 mg. of potassium t-butoxide. After three minutesthe solution is neutralized with acetic acid, evaporated to dryness, andthe residue distributed between chloroform and 5% sodium bicarbonatesolution. The chloroform layer is Washed with water, dried over sodiumsulfate, and concentrated to dryness. The product is recrystallized fromacetone-hexane to give about 14 mg. of A-nor-testosterone, M.P.169-172".

Similarly, 5zx-hydroxy-A-nor 5a androstane-2,20-dione,5u,11u-dihydrOXy-ZOB-acetoxy-A-nor-5a pregnane- 2-one, and511,15a-dihydroxy-ZOB-acetoxy-A-nor-Sa-pregnane-2-one are converted toA-norandrost-3-one-2,20- dione, 11a.hydroxy-2OB-acetoxy-A-norpregn-3-en-2-one, and15a-hydroxy-20fi-acetoxy-A-norpregn-3-en-2-one respectively.

This invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A method of converting a compound selected from the group consistingof a steroid of the A-nor-Sa-andro- :stane series and the A-nor-5x-pregnane series, saturated in both the A and B rings and containing aSet-hydrogen substituent to a 5a-hydroxyl derivative, which comprisessubjecting said steroid to the action of enzymes of Cokeromycesrecurvatus, under oxidizing conditions, and recovering the 5 x-hydroxysteroid formed.

2. The method of claim 1 wherein the steroid is of the A-nOr-Sa-pregnaneseries.

3. The method of claim 1 wherein the steroid is of the2-keto-A-nor-5a-pregnane series.

4. A method for preparing 5a-hydroxy-A-nor-5a-pregnane-2,20-diode, whichcomprises subjecting A-IlOf-5apregnane-2,20-dione to the action ofenzymes of Cokeromyces recurvatus, under oxidizing conditions andrecovering the 5a-hydroxy steroid formed.

5. A method for preparing 5a,l7,8-dihydroxy-A-nor- 5a-androstane-2-one,which comprises subjecting 20,8-hydroxy-A-nor-Sa-pregnane-Z-One to theaction of enzymes of Cokeromyces recurvatus, under oxidizing conditionsand recovering the 5ot-hydroxy steroid formed.

References Cited by the Examiner UNITED STATES PATENTS 3,143,480 8/1964Laskin 5l A. LOUIS MONACELL, Primary Examiner.

ALVIN E. TANENHOLTZ, Examiner.

1. A METHOD OF CONVERTING A COMPOUND SELECTED FROM THE GROUP CONSISTINGOF A STEROID OF THE A-NOR-5A-ANDROSTANE SERIES AND THE A-NOR-5A-PREGNANESERIES, SATURATED IN BOTH THE A AND B RINGS AND CONTAINING A 5A-HYDROGENSUBSTITUENT TO A 5A-HYDROXYL DERIVATIVE, WHICH COMPRISES SUBJECTING SAIDSTEROID TO THE ACTION OF ENZYMES OF COKEROMYCES RECURVATUS, UNEROXIDIZING CONDITIONS, AND RECOVERING THE 5A-HYDROXY STEROID FORMED.